Infertility affects approximately 10% of couples due to a combination of both male and female reproductive disorders. The proportion of infertile men and women is similar, but the etiology of the condition is sex-specific. The project aims to characterize molecular mechanisms required for sperm production. Genetic studies in rodents showed that several RNA binding proteins (RBPs) are necessary at different stages of gametogenesis; however, little is known about their functions mechanistically. We aim to gain insights into the role of RNA metabolism in sperm production by focusing on the role of RNA modifications. RBPs can promote transcript translation, storage or degradation and is the balance between different protein complexes that decides the fate of the mRNA. In the process, the molecular machinery leaves various marks/modifications on the RNA that determine the molecular pathways the transcripts will follow. In particular, the 3 ends of mRNAs are tagged by specialized complexes that can sort the transcript into the different RNA processing compartments. For example, the Terminal Uridylyl-Transferases, TUT4 and TUT7, tag the mRNAs tails with non-templated Us, a process known as uridylation, that leads to transcript decay. We are now depleting TUT4 and TUT7 in different types of male germ cell, where they are highly expressed, to assess the relevance of Us additions to mRNAs in gametogenesis. The most common type of mRNA 3 end modification is the addition of a poly(A) tail. Although polyadenylation is believed to be critical in the germline, little is known about cytoplasmic poly(A) tail dynamics and the molecular machinery responsible for the modification in germ cells. Several Terminal Nucleotidyl Transferases (TENTs) with polyadenylation potential are expressed during spermatogenesis. To evaluate the physiological relevance of the TENTs, we are generating animal models to deplete the proteins at various stages of male gametogenesis. To capture the relevance of 3 addition in the continuous differentiation process of germ cells, we need methods to measure RNA modifications from small populations of cells. To this end, we are improving and developing strategies to increase the sensitivity of methods to measure poly(A) tail length and other 3 end additions. By combining advanced mouse genetics and state of the art molecular biology techniques, we will better understand the mechanisms of RNA metabolism that shape gametogenesis and could lead to infertility when disrupted.